A flow cytometric assay for target binding by NKH1A+ cells using a single laser system.
A modified method of analysis by flow cytometry has been adapted to the NK/target cell system to monitor conjugate formation within a mixed population. The two-parameter assay is based on the use of two colour fluorescent markers in which an indirect FITC-conjugated NKH1A antibody (green fluorescence) is used to detect NK-specific effector cells and an intracellular strain, hydroethidine (red fluorescence) is used to detect target cells. The two cell populations and their conjugates are discernible by the presence of both green and red fluorescence, using a single laser beam tuned to 488 nm. In comparison, a manual count conjugate assay was applied using a hemacytometer and fluorescence microscope. Of the two methods, the automated technique using flow cytometry provides a lower but more precise representation of conjugate formation, due to the avoidance of technical/observer error that is common to manual count assays. The cytometric method has proven to be reproducible and superior in consistency to the manual count assay and can be adapted to almost any effector/target system under investigation. In comparison with previous methods using dual laser, the single laser system provides advantages of cost and time efficiency due to setup simplicity, as well as improved availability.
Zanyk
M J
MJ
Department of Pathology, University of Western Ontario, London, Canada.
Banerjee
D
D
McFarlane
D L
DL
D000911:Antibodies, Monoclonal; D002448:Cell Adhesion / Q000276:immunology; D002469:Cell Separation; D005434:Flow Cytometry / Q000379:methods; D005456:Fluorescent Dyes; D006801:Humans; D007694:Killer Cells, Natural / Q000276:immunology*; D007834:Lasers; D009369:Neoplasms / Q000276:immunology*; D010641:Phenotype; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes